Stereo/regio-selective access to substituted 3-hydroxy-oxindoles with anti-proliferative assessment and in silico validation

The manuscript focuses on a highly stereo-/regioselective approach for synthesizing a diverse array of substituted-3-hydroxy-2-oxindoles. The synthesized compounds were subsequently subjected to anti-proliferative assessment against various cell lines, including colorectal carcinoma, ovarian cancer, and human metastatic melanoma cancer. The structural characterization of the synthesized scaffolds was unambiguously confirmed using X-ray diffraction analysis. Among the synthesized compounds, one compound demonstrated exceptional potency within the series. It exhibited 1.2, 2.12, and 1.55 times greater potency than cisplatin against the HCT116, OVCAR10, and 1205Lu cell lines, respectively. These results were further supported by in vitro caspase-mediated apoptosis studies. Molecular docking studies of these compounds on the target VEGFR2 protein revealed their binding capability.


Introduction
Cancer is the second most common cause of mortality accounting for one in every six fatalities and is challenging to treat.According to data from the American Cancer Society, cancer was predicted to be the cause of nearly 609 820 US fatalities in 2023 which is about 1670 deaths per day. 1 Vascular endothelial growth factor receptor 2 (VEGFR2) has been identied as a key target for anticancer therapy.VEGFR2 is a member of the tyrosine kinase receptor family, and its inhibition prevents angiogenesis by blocking phosphorylation and dimerization. 2,3VEGFR2 is crucial for normal physiological conditions, but its deregulation is associated with cancer. 4Tumour cells produce VEGF, which activates VEGFR2 and stimulates the growth of new blood vessels, encouraging tumour angiogenesis and cancer spread. 5,6nhibiting VEGFR2 has emerged as a potential strategy to halt cancer growth, and various VEGFR2 inhibitors have undergone clinical testing, with some approved for use.VEGFR2 inhibitors, including sorafenib, sunitinib, pazopanib, lenvatinib, vandetanib, and cabozantinib, have been approved for use by the FDA in treating different types of cancers such as thyroid cancer, renal cell carcinoma, and hepatocellular carcinoma. 7,8However, these drugs have limitations such as resistance and side effects, necessitating the identication of newer, more effective scaffolds with fewer side effects.
Isatin (1H-indol-2,3-dione) is a naturally occurring heterocyclic compound found in various plants such as Isatis tinctoria, Couroupita guianensis Aubl, Melochia tomentosa, and Boronia koniamboensis. 9Additionally, it is discovered in the parotid gland of Bufo frogs and Dicathais orbita, an Australian mollusc. 10It is recognised as a tryptophan or epinephrine metabolite in humans and is found in body uids, peripheral tissues, and the central nervous system. 11Isatin offers a versatile platform for structural modication and derivatization due to its molecular architecture.It can take on the roles of an electrophile and a nucleophile.A variety of isatin derivatives have biological properties that include anti-cancer, anti-fungal, anti-HIV, and anti-inammatory, etc. 12 In the realm of organic synthesis, heterocyclic fused threemembered ring system has been crucial.The inherent ring strain of these molecules can be harnessed into ring-opening/ expansion reactions. 13Due to their high reactivity and frequent presence in bioactive small molecules and several natural compounds, epoxides are well-known to both organic and medicinal chemists. 14Spiro-epoxyoxindoles possessing an epoxide ring fused to the C-3 position of the oxindole core are regarded as complex building blocks.The presence of heteroatoms along with inherent ring strain makes them susceptible to nucleophilic ring-opening reactions. 15The potential utility of these privileged frameworks in organic synthesis as well as drug discovery has lured many synthetic as well as medicinal chemists. 16The spiro-epoxyoxindoles have been predominantly known for their nucleophilic ring-opening and Friedel-Cras arylation reactions to give 3,3-disubstituted oxindoles and spiro-oxindoles. 17The 3-substituted-3-hydroxy-2-oxindole scaffold is indeed a signicant structural motif found in several natural compounds with diverse biological activities.This scaffold has garnered attention in the eld of medicinal chemistry due to the potent properties it exhibits including antioxidative, anticancer, anti-HIV, neuroprotective effects, and other biological characteristics. 18There is an expanding list of bioactive 3-substituted-3-hydroxy-2-oxindole natural products, such as convolutamydines, arundaphine, maremycins, CPC-1, etc. as shown in Fig. 1.The 3-substituted-3-hydroxy-2oxindoles have also found their place as targets in the arena of drug discovery in addition to being key intermediates towards complex natural product synthesis.
Our research team recently showed that adding tetrahydro-bcarboline (THbC) to spiro-epoxy oxindoles resulted in 3-THbCsubstituted-3-hydroxy-oxindole with good antiproliferative effects against the MCF-7 (ER+) and MDA-MB-231 (ER) cell lines. 19Building upon previous studies, [20][21][22] this manuscript focuses on the design, synthesis, characterization, and antiproliferative evaluation of scaffolds that incorporate substituted N-benzylated oxindoles with various secondary amines through nucleophilic ring opening of corresponding oxiranes.To compare the effect of the presence of the N-benzyl group with the free -NH group of the isatin core, the corresponding unsubstituted oxindole counterparts have also been synthesised and evaluated.

Synthetic chemistry
The synthesis of 3-substituted 3-hydroxy-oxindole was initiated with the preparation of a precursor spiro-epoxyoxindole, which was achieved through the Corey-Chaykovsky epoxidation reaction.Spiro-epoxyoxindole 3e was obtained by treating isatin with trimethyl sulfoxonium iodide (TMSI) in the presence of sodium hydride and DMF.For the preparation of N-benzylated spiro-epoxyoxindoles 3a-d, a base-promoted benzylation of substituted isatins 1a-d were carried out resulting in the formation of N-benzylated isatins 2a-d.The subsequent treatment of 2a-d with trimethyl sulfoxonium iodide (TMSI) yielded the desired N-benzylated spiro-epoxyoxindoles 3a-d (Scheme 1).
To optimise the conditions of ring opening, a model reaction between spiro-epoxyoxindole 3a and morpholine was carried out in distinct solvents, namely non-polar toluene, polar aprotic tetrahydrofuran (THF), and polar protic carbinol in the presence or absence of a catalytic quantity of p-toluene sulfonic acid (p-TsOH) (Scheme 2).As evident, the presence of p-TsOH as catalyst promoted the reaction specically in case of THF and toluene, primarily by coordinating with the oxirane oxygen and facilitating the ring opening.The opening of oxirane ring resulted in the formation of 4a, the structure of which could either be 4a(I), resulted from nucleophilic ring opening from less hindered site or 4b(II) resulted from ring opening from more hindered site.However, in case of carbinol, the presence of catalytic amount of p-TsOH led to a mixture of products 4a and 5. Compound 5 was formed by the participation of solvent as the nucleophile.In terms of the mechanism, three possible pathways can be proposed for the ring opening in the presence of p-TsOH.The initial protonation of the oxirane ring is facilitated by p-TsOH.Path A involves the formation of a stable carbocation and subsequent ring opening, followed by the attack of morpholine.On the other hand, path B involves the assistance of cyclic amidic oxygen, leading to ring opening.In both cases, the ring opens at a site that is more hindered, resulting in the formation of a thermodynamically stable product 4a(I). 23,24Furthermore, morpholine, being a good nucleophile, can also attack from the less hindered site immediately aer protonation, leading to the formation of a product 4a(II) that is controlled by kinetics, according to path C (Fig. 2).
However, in the absence of p-TsOH, a pseudo S N 2 mechanism can be proposed for the ring opening, which yields a kinetically controlled product.Initially, the solvent promotes the activation of the epoxide ring.The transition state I (TS-I) is stabilized through hydrogen bonding, and subsequently, with the assistance of cyclic amidic oxygen, nucleophilic ring opening occurs from the less hindered site, resulting in the formation of product 4a(II) (Fig. 3).
X-ray diffraction studies were performed to unequivocally establish the structure of 4a.Evidently, the epoxide ring opening proceeded via nucleophilic attack from the less hindered position as conrmed by the crystal structure, shown in Fig. 4. Table 1 provides statistics on crystallographic data and renement.
Furthermore, analytical evidence and spectral data were used to characterise synthesised molecule 4a.It exhibited a molecular ion peak [M] + at 338.1642 in its High Resolution Mass Spectrum (HRMS). 1 H NMR of 4a showed characteristic pair of doublets at d 5.07 and 4.72 with a coupling constant of 15.75 corresponding to benzylic -CH 2 and a multiplet at d 3.67-3.58corresponding to methylene groups of morpholine -CH 2 -O-CH 2 .The absorption peaks at d 177.46 and 108.35 in the 13 C NMR spectrum, corresponding to carbonyl and methine groups of isatin further validated the assigned structure.
Having determined the suitable conditions, the desired scaffolds, 3-substituted 3-hydroxy-oxindoles were synthesized by heating the spiro-epoxyoxindoles with various secondary amines viz.morpholine/piperidine/pyrrolindine using carbinol as a solvent for 2 h at 60 °C (Scheme 3).The crude products thus obtained were puried via column chromatography using ethyl acetate/hexane (85 : 15) as an eluent.In vitro studies Biological evaluation and SAR.The newly synthesized compounds were subjected to assessment for their potential to inhibit the proliferation of various cancer cell lines including colorectal carcinoma (HCT116), ovarian cancer (OVCAR 10), and human metastatic melanoma (1205Lu), and non-cancerous fetal human cells (FHC).The MTS assay was employed, and the resulting IC 50 values are tabulated in Table 2.It is evident that the majority of the synthesized compounds did not demonstrate noteworthy cytotoxic effects against the tested cell lines.However, one compound, 4j, displayed remarkable inhibitory activities across all tested cell lines.Notably, compound 4j exhibited an IC 50 value of 9.63 mM, rendering it particularly effective against the HCT116 cell line.This efficacy surpassed that of the reference drugs 5-uorouracil and cisplatin, which showed IC 50 values of 22.26 mM and 12.11 mM, respectively.Additionally, 4j emerged as the most potent analog in the series against the OVCAR 10 cell line, with an IC 50 value of 8.56 mM, outperforming cisplatin (IC 50 = 18.22 mM) and 5-uorouracil (IC 50 = 22.4 mM).Furthermore, in comparison to 5-uorouracil (IC 50 = 15.4 mM) and cisplatin (IC 50 = 19.01 mM), compound 4j displayed signicant effectiveness against the 1205Lu cell line, exhibiting an IC 50 value of 12.22 mM.In order to ascertain whether compound 4j is selectively cytotoxic or antiproliferative to cancer cells, it was tested for non-cancerous fetal human cells (FHC).The compound displayed acceptable cytotoxicity towards FHC with an IC 50 of 20.56 mM which was comparable to that of standards 5-FU and cisplatin having IC 50 values of 21.3 and 25.13 mM, respectively.
In summation, while the majority of synthesized compounds showed limited impact on the tested cell lines, compound 4j demonstrated impressive and consistent efficacy across all cell lines, making it a promising candidate for further investigation as an antiproliferative agent.
Moreover, 4j demonstrated a superior selectivity index (SI) across all the tested strains enlisted in Table 3.The SI was determined for each compound by employing the formula: SI = (IC 50 for normal cell line FHC)/(IC 50 for respective cancerous cell line).A favourable SI value > 1.0 signies that the drug is more effective against tumor cells compared to its toxicity on normal cells.Comparing 4j to standard 5-FU and cisplatin, it exhibits good selectivity for HCT116, OVCAR10, and 1205Lu cancer cell lines, respectively, with SI values of 2.12, 2.40, and 1.68.
The inhibition data presented in Table 2 was used to derive the following structure-activity relationship (SAR): (1) The presence of the benzyl group at the N-1 of the isatin core improved anti-proliferative activities as evidenced by the lack of activities of N-unsubstituted compounds 4m-o.(2) The presence of Br at C-7 of the isatin core is preferred over H in the case of morpholine-substituted oxindole while negligible effects are observed in the case of piperidine-/ pyrrolidine-substituted oxindoles.
(3) In the case of morpholine-substituted oxindoles, the antiproliferative activities follow the order: di-Br > Br > H = Cl.The order gets reversed in the case of piperidine/pyrrolidinesubstituted oxindoles and the pattern followed is H > Br > Cl = di-Br.
The graphical representation of SAR is illustrated in Fig. 5.
Apoptosis assay.Apoptosis, a carefully regulated and anatomically distinct form of cell suicide, is a mechanism used by multicellular organisms to get rid of undesired, damaged, or diseased cells.Apoptosis is essential for sustaining adult tissues as well as for embryonic development, and its dysregulation can lead to autoimmune and neurological illnesses as well as cancer.Caspases, which may be activated by two distinct signalling routes are cysteine proteases that cleave aer aspartate residues, cause apoptosis.There are two pathways at play: the intrinsic pathway, which is started by stress or damage inside the cell, and the extrinsic pathway, which is started extracellularly by the ligation of "death receptors," a subset of the tumour necrosis factor (TNF) receptor complex.TNF-related apoptosisinducing ligand (TRAIL) is a viable possibility for targeted cancer treatment because, in contrast to other TNF family ligands like CD95l, it may specically cause apoptosis in tumour cells. 25,26ased on cell viability data, 4j showed signicant antiproliferative activity against the tested cancer cells.We further carried out apoptosis experiments utilizing the caspase-3/7 7-AAD and Annexin V/7-AAD assays to evaluate the mode of cell death.As seen in Fig. 6, 10 mM of 4j was used for HCT-116 cells treatment for 24 and 48 h before the cells underwent apoptotic experiments.The data showed a signicant increase in the apoptotic cell population aer the treatment with 4j.In the Annexin V/7-AAD experiment, 4j treated HCT-116 cells showed 19.5% of apoptotic cells at 24 h and 46.15% at 48 h treatment.Similar to this, in HCT-116 cells treated with 4j for 24 and 48 h showed 21.5% and 57.9% of the cell population were found to be apoptotic by the Caspase 3/7 7-AAD assay, respectively.The results demonstrated that 4j has a stronger cytotoxic effect on HCT-116 cells which is mediated through the caspase apoptotic pathway.
In silico studies Molecular docking analysis.We conducted in silico analyses employing three compounds-4j (active), 4k (inactive), and 5-uorouracil (reference)-against the VEGFR2 protein (PDB ID: 3VHE) to explain the observed anticancer activity and locate the unique binding core.The results of the docking tests showed that 4j had a good binding mode and was successfully docked inside the protein's active site as shown in Fig. 7. Further, the docking calculations of 4j, 4k and 5-uorouracil are summarised in Table 4.The docking score for 4j was observed to be −6.228,whereas for 4k and the reference drug, it was found to be −3.857 and −4.198, respectively.
During studies two distinct types of interactions were iden-tied.For both 4j and 4k compounds, an induced dipole-dipole   interaction occurred between C6 and ASP814, alongside a hydrogen bonding interaction between -OH and ASP1046.Notably, in the case of 4j, an additional polar interaction was detected involving the oxygen within the morpholine group and the adjacent polar group, SER885, which is located in the active site.This specic interaction was absent in the case of 4k.The stronger protein-ligand interactions and a higher docking score for 4j compared to 4k provided support for the enhanced cytotoxic activity of 4j.Thus, from the analyses of molecular docking data, it can be inferred that the active compound 4j outperforms the reference drug, 5-uorouracil with notable protein interactions.Consequently, the suggested mechanism of action for 4j likely correlates with its cytotoxic effects against the VEGFR2 protein.
ADMET analysis.ADMET is an acronym that represents the vital processes of Absorption, Distribution, Metabolism, Excretion, and Toxicity.These processes play a pivotal role in determining a drug's pharmacokinetics and pharmacodynamics.Understanding these aspects is crucial for comprehending a drug's behavior within the body and assessing its effectiveness and safety.The ADMET descriptor module of small molecules was utilized for ADMET prediction, accessible at http:// www.swissadme.ch/ and http://biosig.unimelb.edu.au/pkcsm/prediction.The acquired values are presented in Table S1 of the ESI.† The ADMET studies for compounds 4a, 4d, 4j and 4m predicted that the number of rotatable bonds, hydrogen bond acceptors, and donors, and a total polar surface area lies within the recommended range with no violation of the Lipinski's rule of ve.Among these, compounds 4a, 4d and 4m displayed log P o/w values below 3, while compound 4j exhibited log P o/w value above 3 suggesting 4j possessed noteworthy permeability through the cell membranes, indicating a well-balanced interplay between hydrophobic and hydrophilic traits highlighting its potential as a promising drug candidate.The compounds 4a and 4d were predicted to be P-glycoprotein II inhibitor while compound 4j was predicted to be both P-glycoprotein I and Pglycoprotein II inhibitor indicating good absorption and permeability.All the three compounds 4a, 4d and 4j can act as P-glycoprotein substrates while 4m cannot.Furthermore, all compounds showed high human intestinal absorption, indicating high oral bioavailability and affinity for interacting with target cells.The log BB (blood-brain barrier permeability) values suggested that these compounds exhibited substantial potential to cross the blood-brain barrier and enter the central nervous system (CNS).In conclusion, the ADMET studies predicted good oral absorption, permeability, and excellent intestinal bioavailability for the compounds.

Experimental
General information 1 H NMR and 13 C NMR spectra were recorded in deuterochloroform (CDCl 3 ) and dimethylsulfoxide (DMSO-d 6 ) with Jeol 400 (400 MHz) and Bruker 500 (500 MHz) spectrometer using TMS as an internal standard.Chemical shi values are expressed as parts per million downeld from TMS and J values are in hertz.Splitting patterns are indicated as s: singlet, d: doublet, t: triplet, m: multiplet, dd: double doublet, ddd: doublet of a doublet of a doublet, and br: broad peak.High resolution mass spectra were recorded on a Bruker-micrOTOF-Q II spectrometer.
General procedure for synthesis of C-5 substituted N-benzylated isatin 2(a-d).To a stirred solution of substituted isatin 1(a-d) (1 mmol) in anhydrous DMF, sodium hydride (1.3 mmol) was added.The reaction mixture was allowed to stir for half an hour at 0 °C.The change of color from orange to dark purple conrmed the formation of anionic intermediate, subsequently benzyl bromide (1.2 mmol) was added to the reaction mixture.Reaction mixture was then allowed to stir for 1 h.Completion of  Paper RSC Advances the reaction was conrmed by TLC.Aer the completion of reaction, ice was added to the reaction mixture resulting in the precipitation of product from reaction mixture.The product was then ltered and dried to get 2(a-d) in good yields (not here).The crude product mixture was then puried by column chromatography on silica gel (60-120 mesh) using ethyl acetate : hexane (15 : 85) mixture as the eluent to afford the pure product.
General procedure for synthesis of spiro[indoline-3,2 ′oxiran]-2-one (3e) and 1-benzylspiro[indoline-3,2 ′ -oxiran]-2-one 3(a-d).A mixture of trimethylsulfoxonium iodide (1.2 mmol) and sodium hydride (1.0 mmol) in dry DMF was stirred at 0 °C for 30 minutes.Isatin (1.0 mmol) in dry DMF (5 mL) as added dropwise to this solution over 10 minutes.The progress of the reaction was monitored by TLC.Aer completion of the reaction, ice was added to the reaction mixture resulting in the precipitation of the desired product from the reaction mixture.The product was then ltered and dried to get 3e in good yields.Similarly, N-benzylisatin (1.0 mmol) in dry DMF (5 mL) as added dropwise to the solution of Trimethylsulfoxonium iodide (1.2 mmol) and sodium hydride (1.0 mmol) in dry DMF over 10 minutes.The progress of the reaction was monitored by TLC.Aer completion of the reaction, ice was added to the reaction mixture resulting in the precipitation of the desired product from the reaction mixture.The product was then ltered and dried to get 3(a-d) in good yields.
General procedure for synthesis of 3-substituted-3-hydroxy-2-oxindoles derivatives 4(a-o).To a well stirred solution of spiro-epoxy isatin 3(a-d) (1.0 mmol) in dry carbinol, secondary amines (piperidine/morpholine/pyrrolidine) (2.0 mmol) were added and the reaction mixture was stirred for 2 h.Upon completion of reaction, as monitored by TLC, solvent was removed under reduced pressure.The reaction mixture was extracted with ethyl acetate (3 × 10 mL).Combined organic layers were dried over anhydrous sodium sulphate and concentrated under reduced pressure.The reaction mixture was puried via column chromatography using ethyl acetate : hexane (40 : 60) to yield target compounds 4(a-o) in appreciable yields.

Apoptosis assays
The apoptosis assay using Muse Annexin V/7-AAD and Caspase 3/7 7-AAD assay kit (EMD Millipore, Darmstadt, Germany) was performed following manufacturer's protocol as described earlier. 28Briey, the cells were seeded in 6-well plate at a density of 1 × 10 5 cells per well.Aer 24 h incubation, the cells were treated with 10 mM of compound 4j.The cells were incubated for 24 and 48 h.At the end of the treatment period, the cells were harvested using enzyme free cell dissociation buffer.The samples were treated with the respective dyes based on the manufacturer's protocol.The samples were further analysed in Muse Cell Analyzer (Merck Millipore, Darmstadt, Germany).

Single crystal XRD
Single crystals of C 20 H 22 N 2 O 3 .A suitable crystal were selected for indexing and the intensity data were recorded at 296 K on a Bruker DUO diffractometer equipped with a Mo Incoatec Microfocus Source ImS (l = 0.71073 Å).Using Olex2, 29 the structure was solved with the XS structure solution program using Direct Methods and rened with the XL 30 renement package using Least Squares minimisation.All non-hydrogen atoms were rened anisotropically and all H atoms linked to N and O atoms were located from the electron density difference map and rened riding on their parent atoms.CCDC-2289774 (for 4a) contain the supplementary crystallographic data for this paper.†

Molecular docking protocols
The molecular docking was performed using Schrodinger (Schrödinger Release 2022-1: Maestro, Schrödinger, LLC, New York, NY).Crystal co-ordinates of VEGFR2 protein (PDB ID: 3VHE) were downloaded from the protein data bank (https:// www.rcsb.org).In the rst step, bond orders were assigned and hydrogens were added by pre-process option.All water molecules were deleted.The heteroatoms are ionized by epic at biological pH to consider the protein permeability and drug solubility and then the H-bonds were optimized to reduce the steric clashes by histidine, aspartate, glutamate, and hydroxyl containing amino acids.Then complete protein structure was minimized by using OPLS 2005 force eld.Ligand preparation is generally required because molecules lack 3D coordinates, ionization, stereochemistry and tautomers.Thus, before docking, the least energy state of ligand was needed to be prepared.Ligprep tool of Schrödinger was used to prepare the least energy state of ligand which converts 1D/2D structures to 3D.Finally, this ligand molecule was minimized with the help of the OPLS 2005 force eld.For docking, the grids were generated by using the grid-based energy descriptor which had a default set of options with van der Waals radius of 1.0.The glide docking of the molecules was carried out using the previously prepared receptor grid and the ligand molecules.The favourable interactions between ligand molecules and the receptor were scored using Glide ligand docking program.All the docking calculations were performed using extra precision (XP) mode and OPLS-2005 force eld.[33][34][35]

Conclusion
The present manuscript involves the synthesis of 3-substituted-3-hydroxy-2-oxindoles derivatives and their antiproliferative evaluation against HCT116, OVCAR 10, 1205Lu, and FHC cell lines.Among the synthesized compounds, morpholinesubstituted di-bromo derivative 4j showed promising activity against all the tested cancer cell lines.With the IC 50 values of 9.63 and 8.56 mM against HCT116 and OVCAR, respectively, it outperforms the 5-FU and cisplatin.The activity in the case of 1205Lu (IC 50 = 12.22 mM) was also better than the reference drugs used.Furthermore, 4j demonstrated an increased induction of apoptotic cell death in the HCT116 cell line, thus conrming the involvement of caspase-mediated apoptotic mechanisms.Molecular docking studies shed light on the mode of action, revealing that the active compound surpasses the reference drug, 5-uorouracil through notable protein interactions.These interactions are implicated in its cytotoxic effects, wherein binding with the VEGFR2 protein is particularly signicant.

Fig. 2
Fig. 2 Possible mechanism in the presence of non-polar/polar aprotic solvent in the presence of p-TsOH.

Fig. 3
Fig. 3 Possible mechanism in presence of polar protic solvent in absence of p-TsOH.

Fig. 6
Fig. 6 To assess the mode of cell death caused by compound 4j, apoptosis assays were performed.(A) The HCT-116 cells were treated with 10 mM of compound 4j for 24 and 48 h and subjected to Annexin V/7-AAD assay and Caspase 3/7 assays.The quantitative assessment of the apoptosis assays is presented as a bar graph in (B) and (C).

Table 1
Crystal data and structure refinement for 4a calc /g cm−3

Table 3
The SI values of the synthesized compounds against HCT116, OVCAR10, 1205Lu, and FHC cell lines